Increased human travel, urbanization and climate changes have resulted in a wide geographic spread of febrile illness caused by both Dengue Virus (DENV) and Chikungunya Virus (CHIKV). These two infections show a substantial overlap in clinical presentation. In addition, both are also co-prevalent in many countries. This poses a significant challenge to clinicians, as the two illnesses require different clinical management. A recent infection can only be confirmed by detection of the virus in a blood sample, as positive results in serological assays may instead be indicative of prior infection. However, as antibodies develop in response to the infection, the viral load in the blood decreases. Thus, sensitive and specific detection of these viruses is imperative for an accurate diagnosis. Typically, molecular methods that employ reverse transcriptase real-time polymerase chain reaction (rtRT-PCR) based detection of viral RNA require nucleic acid extraction of whole blood samples. In this Phase I plan, we propose to (1) develop a multiplexed molecular assay for the direct detection of either DENV or CHIKV in blood samples, and then to (2) incorporate this multiplexed assay into a Collect-to-Test (C2T) system in order to overcome the need for complex sample processing. The C2T system includes a simple blood collection and processing method that requires no pipetting or sample manipulation, combined with a cartridge containing the dried-down assay reagents for simultaneous sample preparation and template amplification. The sample preparation and cartridge loading are equivalent to methods currently used to run lateral flow devices. This system will allow for simultaneous detection of DENV and CHIKV by rtRT-PCR from a minimal volume of whole blood (i.e., a finger prick) without the need for centrifugation or time- consuming extraction methods. Critical to this is the development of a rtRT-PCR-based assay optimized for the detection of viral RNA directly from blood samples and requiring no further sample extraction. These reagents, together with the C2T system, will be compatible with the T- COR 8 portable thermocycler. Tetracore has extensive experience in the development of highly sensitive and specific dried-down, room temperature-stable assays for real-time PCR, including a DENV-specific assay previously developed with Naval Medical Research Center. This assay was systematically evaluated and successfully tested using clinical samples on a commercial laboratory rtRT-PCR platform. Based on our preliminary data and experience we are confident to propose this plan to develop a multiplex rtRT-PCR assay for differential detection of DENV and CHIKV that can be combined for use with the C2T cartridge and the T-COR 8 portable thermocycler, to create an integrated point-of-care system that can be equally effectively in both public health labs and low resource settings.